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Fig. 1. Reversion of committed pOCs by blocking JNK activity. (A) Experimental scheme of osteoclastogenesis from mouse BMMs. Non-adherent bone marrow cells (BMCs) were cultured with M-CSF (30 ng/ml) for 3 days to generate BMMs. BMMs were cultured with M-CSF (30 ng/ml) plus RANKL (100 ng/ml) for 2 days to generate pOCs. (A-C) pOCs were incubated for 48 hours with the NF-
B inhibitor SN50 (SN, 20 µM), JNK inhibitor SP600125 (SP, 10 µM), MEK inhibitor PD98059 (PD, 10 µM), p38 inhibitor SB203580 (SB, 10 µM) or the vehicle (Vh, 0.1% DMSO) in the presence of M-CSF (30 ng/ml) and the indicated combination of RANKL (R, 100 ng/ml), TNF
(T, 20 ng/ml) and IL1 (I, 10 ng/ml). Cells were stained and the number of TRAP+ MNCs was counted. (A,D-F) pOCs were incubated for 24 hours with the inhibitor as above in the presence of M-CSF and RANKL (+MR). Cells were either stained for TRAP (D,E) or incubated with CCK-8 (F) to assess the percentage of TRAP+ cells and the viability of cells, respectively. (A,G) pOCs were treated for 24 hours with SP600125 at the indicated concentration (G) together with M-CSF and RANKL. (A,H) pOCs were incubated for 24 hours with or without SP600125 (10 µM) in the presence of M-CSF and RANKL, and mRNA levels of TRAP, calcitonin receptor (CTR) and MMP9 were analyzed by real-time PCR. (C,E-H) Error bars represent s.d. from triplicate samples. Similar results were obtained in two other experiments. txt, treatment.
